I. Field of the Invention
This invention relates to a method of culturing a protoplast. More specifically, this invention relates to a method of culturing a protoplast in a liquid medium, by which a cell cluster or a callus is derived from the protoplast.
II. Description of the Prior Art
A protoplast is a cell of a plant, bacterium, fungus or the like from which the cell wall has been removed. Since the protoplast does not have a cell wall, it is easily subjected to an artificial manipulation such as cell fusion, gene manipulation and artificial somatic cell mutation. Thus, if a complete plant can be regenerated from a manipulated protoplast it would be possible to obtain a plant which has an advantageous characteristic which the wild type plant does not have. It is known for many plants that a complete plant can be regenerated from a callus or a cell cluster. Thus, if a callus can be derived from a protoplast, a complete plant is likely to be regenerated from the callus, and in turn, from the protoplast.
Some techniques are known for dicotyledons such as tobacco by which a complete plant can be regenerated from a protoplast. However, as for the gramineous plants such as rice, wheat and corn, complete plants were reported to be regenerated only for corn and pasture. As to rice, very few techniques have been reported as mentioned below. The conventional culturing methods of the protoplasts include culturing the protoplasts by embedding the protoplast in a semi-solid agar medium, by suspending the protoplast in a liquid medium, and by culturing the protoplast using feeder cells. However, it has been found that these techniques are often not effective for culturing other plants such as gramineous plants including rice, wheat and corn. For example, if a protoplast of rice is cultured by one of these methods, the protoplast dies or cannot grow.
As for culturing techniques of the protoplast of rice, it has been reported that a callus was derived from a protoplast obtained from a cell lacking its nitrate reductase (Wakasa et al., J. Plant Physiol. 117: pp. 223-231, (1984)), and that a shoot was generated from a callus derived from a protoplast obtained from a callus of a pollen (Ohno et al., Japanese Journal of Breeding 35: pp.54-55, (1985)). However, these techniques utilize protoplasts released from specific calli, and the techniques are applicable to not all kinds of protoplasts released from various kinds of calli or tissues. In other words, these techniques are not reproducible for most kinds of protoplasts.
On the other hand, it has been reported by many researchers that complete plants were regenerated from cultured cells of rice (Nishi et al., Nature 219: pp. 508-509, (1968)). However, these techniques do not utilize the protoplast. Further, it has been found that obtaining a protoplast from cells having a high differentiation ability used in these techniques is difficult, and to culture the protoplast is also difficult.
Thus, a method of culturing protoplasts is needed to be established by which a callus or a cell cluster is derived from the protoplast, which method is reproducible and applicable to protoplasts originated from a general or a non-specific cell of a plant.